The tubulysins, first isolated by the Höfle/Reichenbach group from myxobacterial cultures (F. Sasse, H. Steinmetz, G. Höfle, H. Reichenbach, J. Antibiot. 2000, 53, 879-885), are exceptionally potent cell-growth inhibitors that act by inhibiting tubulin polymerization and thereby induce apoptosis. (M. W. Khalil, F. Sasse, H. Lünsdorf, Y. A. Elnakady, H. Reichenbach, ChemBioChem, 2006, 7, 678-683; and G. Kaur, M. Hollingshead, S. Holbeck, V. Schauer-Vuka{hacek over (s)}inović, R. F. Camalier, A. Dömling, S. Agarwal, Biochem. J. 2006, 396, 235-242). The tubulysins, of which tubulysin D is the most potent, have activity that exceeds all almost all other tubulin modifiers including, the epothilones, vinblastine, and paclitaxel (Taxol), by 20- to 1000-fold. (H. Steinmetz, N. Glaser, E. Herdtweck, F. Sasse, H. Reichenbach, G. Höfle, Angew. Chem. 2004, 116, 4996-5000; H. Steinmetz, N. Glaser, E. Herdtweck, F. Sasse, H. Reichenbach, G. Höfle, Angew. Chem. Int. Ed. 2004, 43, 4888-4892; and G. Höfle, N. Glaser, T. Leibold, U. Karama, F. Sasse, H. Steinmetz, Pure and Applied Chemistry 2003, 75,167-178). Paclitaxel and vinblastine are current treatments for a variety of cancers, and epothilone derivatives are under active evaluation in clinical trials. Synthetic derivatives of tubulysin D would provide essential information about the mechanism of inhibition and key binding interactions, and could have superior properties as anticancer agents either as isolated entities or as chemical warheads on targeted antibodies or ligands.
Tubulysin D (1) is a complex tetrapeptide that can be divided into four regions as shown in Formula I: Mep (D-N-methyl pipecolinic acid), Ile (L-isoleucine), Tuv (tubuvaline), and Tup (tubuphenylalanine). All of the more potent derivatives of tubulysin, including tubulysin D, also incorporate the interesting O-acyl N,O-acetal functionality, which has rarely been observed in natural products. This reactive functionality is documented to be quite labile to both acidic and basic reaction conditions, and therefore may play a key role in the function of the tubulysins. (J. Iley, R. Moreira, T. Calheiros, E. Mendes, Pharm. Res. 1997, 14, 1634-1639).

Recently, the total synthesis of tubulysin D was reported, which represents the first synthesis of any member of the tubulysin family that incorporates the O-acyl N,O-acetal functionality. (H. M. Peltier, J. P. McMahon, A. W. Patterson, J. A. Ellman, J. Am. Chem. Soc. 2006, 128, 16018-16019).
A cost-efficient, scalable method for the synthesis of tubulysin and tubulysin analogues would be a significant addition to the array of available chemistries. Furthermore, tubulysin analogues that are structurally more simple and approximately as bioactive as the naturally occurring tubulysins would provide for ease of access to important cell growth inhibitors. The current invention addresses this and other needs.